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The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G 1 -, S- or G 2 -phase checkpoints, that ATR is only partly involved in the regulation of S- and G 2 -phase checkpoints and that DNA-PKcs is not involved in checkpoint regulation. However, further analysis of the contributions of these kinases to checkpoint responses in cells exposed to ionizing radiation (IR) recently uncovered striking integrations and interplays among ATM, ATR and DNA-PKcs that adapt not only to the phase of the cell cycle in which cells are irradiated, but also to the load of DNA double-strand breaks (DSBs), presumably to optimize their processing. Specifically, we found that low IR doses in G 2 -phase cells activate a G 2 -checkpoint that is regulated by epistatically coupled ATM and ATR. Thus, inhibition of either kinase suppresses almost fully its activation. At high IR doses, the epistatic ATM/ATR coupling relaxes, yielding to a cooperative regulation. Thus, single-kinase inhibition suppresses partly, and only combined inhibition suppresses fully G 2 -checkpoint activation. Interestingly, DNA-PKcs integrates with ATM/ATR in G 2 -checkpoint control, but functions in its recovery in a dose-independent manner. Strikingly, irradiation during S-phase activates, independently of dose, an exclusively ATR-dependent G 2 checkpoint. Here, ATM couples with DNA-PKcs to regulate checkpoint recovery. In the present work, we extend these studies and investigate organization and functions of these PI3-kinases in the activation of the G 1 checkpoint in cells irradiated either in the G 0 or G 1 phase. We report that ATM is the sole regulator of the G 1 checkpoint after exposure to low IR doses. At high IR doses, ATM remains dominant, but contributions from ATR also become detectable and are associated with limited ATM/ATR-dependent end resection at DSBs. Under these conditions, only combined ATM + ATR inhibition fully abrogates checkpoint and resection. Contributions of DNA-PKcs and CHK2 to the regulation of the G 1 checkpoint are not obvious in these experiments and may be masked by the endpoint employed for checkpoint analysis and perturbations in normal progression through the cell cycle of cells exposed to DNA-PKcs inhibitors. The results broaden our understanding of organization throughout the cell cycle and adaptation with increasing IR dose of the ATM/ATR/DNA-PKcs module to regulate checkpoint responses. They emphasize notable similarities and distinct differences between G 1 -, G 2 - and S-phase checkpoint regulation that may guide DSB processing decisions.

Several studies have demonstrated that mitochondria are critically involved in the pleiotropic manifestation of radiation effects. While conventional whole-cell irradiation compromises the function of mitochondria, the effects of subcellular targeted radiation are not yet fully understood. In this study, normal human diploid cells with cell-cycle indicators were irradiated using a synchrotron X-ray microbeam, and mitochondrial membrane potential was quantified by JC-1 over the 72-h period postirradiation. Cytoplasmic irradiation was observed to temporarily enlarge the mitochondrial area with high membrane potential, while the total mitochondrial area did not change significantly. Unexpectedly, cell-nucleus irradiation promoted a similar increase not only in the mitochondrial areas with high membrane potential, but also in those with low membrane potential, which gave rise to the apparent increase in the total mitochondrial area. Augmentation of the mitochondrial area with low membrane potential was predominantly observed among G1 cells, suggesting that nucleus irradiation during the G1 phase regulated the mitochondrial dynamics of the cytoplasm, presumably through DNA damage in the nucleus., (©2020 by Radiation Research Society. All rights of reproduction in any form reserved.)

Understanding the mitotic DNA damage response (DDR) is critical to our comprehension of cancer, premature aging and developmental disorders which are marked by DNA repair deficiencies. In this study we use a micro-focused laser to induce DNA damage in selected mitotic chromosomes to study the subsequent repair response. Our findings demonstrate that (1) mitotic cells are capable of DNA repair as evidenced by DNA synthesis at damage sites, (2) Repair is attenuated when DNA-PKcs and ATM are simultaneously compromised, (3) Laser damage may permit the observation of previously undetected DDR proteins when damage is elicited by other methods in mitosis, and (4) Twenty five percent of mitotic DNA-damaged cells undergo a subsequent mitosis. Together these findings suggest that mitotic DDR is more complex than previously thought and may involve factors from multiple repair pathways that are better understood in interphase., Competing Interests: The authors have declared that no competing interests exist.

Background Management of locoregionally recurrent head and neck squamous cell carcinomas (HNSCC) is challenging due to potential radioresistance. Pulsed low-dose rate (PLDR) irradiation exploits phenomena of increased radiosensitivity, low-dose hyperradiosensitivity (LDHRS), and inverse dose-rate effect. The purpose of this study was to evaluate LDHRS and the effect of PLDR irradiation in isogenic HNSCC cells with different radiosensitivity. Materials and methods Cell survival after different irradiation regimens in isogenic parental FaDu and radioresistant FaDu-RR cells was determined by clonogenic assay; post irradiation cell cycle distribution was studied by flow cytometry; the expression of DNA damage signalling genes was assesed by reverse transcription-quantitative PCR. Results Radioresistant Fadu-RR cells displayed LDHRS and were more sensitive to PLDR irradiation than parental FaDu cells. In both cell lines, cell cycle was arrested in G2/M phase 5 hours after irradiation. It was restored 24 hours after irradiation in parental, but not in the radioresistant cells, which were arrested in G1-phase. DNA damage signalling genes were under-expressed in radioresistant compared to parental cells. Irradiation increased DNA damage signalling gene expression in radioresistant cells, while in parental cells only few genes were under-expressed. Conclusions We demonstrated LDHRS in isogenic radioresistant cells, but not in the parental cells. Survival of LDHRS-positive radioresistant cells after PLDR was significantly reduced. This reduction in cell survival is associated with variations in DNA damage signalling gene expression observed in response to PLDR most likely through different regulation of cell cycle checkpoints.

Existing evidence has highlighted the effect of ultraviolet light radiation leading to corneal epithelium impairment. During this study, we aim to investigate the effect of microRNA-129-5p (miR-129-5p) on the wound healing process of corneal epithelial cells (CECs) induced by ultraviolet rays in mice by targeting epidermal growth factor receptor (EGFR). First, mouse models of ultraviolet ray-induced CEC injury were established and intrastromally injected with different mimic, inhibitor, and short interfering RNA (siRNA) to detect the effect of miR-129-5p on CEC injury. Subsequently, the corneal tissues were obtained to detect the antioxidant ability and EGFR-positive expression rate. The dual-luciferase reporter gene assay was used to test whether EGFR could directly target miR-129-5p. To further investigate the specific mechanism of miR-129-5p and EGFR in CEC injury, CECs were cultured and transfected with miR-129-5p mimic, miR-129-5p inhibitor, siRNA-EGFR, and miR-129-5p inhibitor + siRNA-EGFR. miR-129-5p has been proven to directly target EGFR. Inhibition of miR-129-5p is able to increase the antioxidant capacity, EGFR-positive rate and the expressions of EGFR, B-cell lymphoma-2, zonula occluden-1, occludin, and keratinocyte growth factor-2, but decrease the expression of vascular endothelial growth factor, BCL2-associated X protein, interleukin (IL)-1β, and IL-4. Inhibition of miR-129-5p arrests cells at the S and G2 phases and decreases apoptosis. Our study provides evidence stating that inhibiting miR-129-5p and upregulating EGFR could aid in the repair of mice CEC injury induced by ultraviolet radiation. Therefore, inhibition of miR-129-5p might provide a basic theory in the repair of CEC injury caused by ultraviolet rays., (© 2018 Wiley Periodicals, Inc.)

Histone posttranslational modifications regulate diverse nuclear functions, including DNA repair. Here, we use mass spectrometry, western blotting, immunohistochemistry and advanced confocal microscopy in order to show radiation-specific changes in the histone signature. We studied wild-type mouse embryonic stem cells (mESCs) and mESCs with a depletion of histone deacetylase 1 (HDAC1), which plays a role in DNA repair. Irradiation by γ-rays increased the S139 phosphorylation of histone H2AX but reduced the level of the H3K9-R17 peptide, which contains S10 phosphorylation (H3S10ph). On an individual cellular level, H3S10ph was low in highly γH2AX-positive UV laser-induced DNA lesions, and this nuclear distribution pattern was not changed by HDAC1 depletion. Despite this fact, spontaneous γH2AX-positive DNA lesions colocalized with large H3S10ph-positive nuclear bodies that appear in the G2 phase of the cell cycle. Similarly, by FLIM-FRET analysis, we observed an interaction between H3S10ph and γH2AX in the G2 phase. However, this interaction was reduced when cells were exposed to γ-rays. A mutual link between H3S10ph and γH2AX was not observed in the G1 phase of the cell cycle. Together, our data show that despite the fact that H3S10ph is not directly involved in DNA repair, a decrease in H3S10 phosphorylation and weakened interaction between H3S10ph and γH2AX is a result of radiation-induced damage of the genome. In this case, γ-irradiation also decreased the number of cells in the G1 phase, characterized by no interaction between H3S10ph and γH2AX., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Cdt1 is rapidly degraded by CRL4 Cdt2 E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within ∼15 min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for ∼30 min in NER-deficient XP-A cells, but was degraded within ∼60 min. The delayed degradation was also dependent on PCNA and CRL4 Cdt2 . The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4 Cdt2 to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.

Unrepaired DNA double-strand breaks (DSBs) induced by ionizing radiation are associated with lethal effects and genomic instability. After the initial breaks and chromatin destabilization, a set of post-translational modifications of histones occurs, including phosphorylation of serine 139 of histone H2AX (γH2AX), which leads to the formation of ionizing radiation-induced foci (IRIF). DSB repair results in the disappearance of most IRIF within hours after exposure, although some remain 24h after irradiation. Their relation to unrepaired DSBs is generally accepted but still controversial. This study evaluates the frequency and kinetics of persistent IRIF and analyzes their impact on cell proliferation. We observed persistent IRIF up to 7 days postirradiation, and more than 70% of cells exposed to 5Gy had at least one of these persistent IRIF 24h after exposure. Moreover we demonstrated that persistent IRIF did not block cell proliferation definitively. The frequency of IRIF was lower in daughter cells, due to asymmetric distribution of IRIF between some of them. We report a positive association between the presence of IRIF and the likelihood of DNA missegregation. Hence, the structure formed after the passage of a persistent IRI focus across the S and G2 phases may impede the correct segregation of the affected chromosome's sister chromatids. The ensuing abnormal resolution of anaphase might therefore cause the nature of IRIF in daughter-cell nuclei to differ before and after the first cell division. The resulting atypical chromosomal assembly may be lethal or result in a gene dosage imbalance and possibly enhanced genomic instability, in particular in the daughter cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5-8 Gy gamma-ray irradiation delivered to synchronized cells during the G 0 /G 1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single substitution in the T2609 cluster abolished SCE formation and thus also effectively interferes with HRR.

Our recent study showed that quiescent G0 cells are more resistant to ionizing radiation than G1 cells; however, the underlying mechanism for this increased radioresistance is unknown. Based on the relatively lower DNA damage induced in G0 cells, we hypothesize that these cells are exposed to less oxidative stress during exposure. As a catalytic subunit of NADPH oxidase, Ras-related C3 botulinum toxin substrate 2 (RAC2) may be involved in the cellular response to ionizing radiation. Here, we show that RAC2 was expressed at low levels in G0 cells but increased substantially in G1 cells. Relative to G1 cells, the total antioxidant capacity in G0 phase cells increased upon exposure to X-ray radiation, whereas the intracellular concentration of ROS and malondialdehyde increased only slightly. The induction of DNA single- and double-stranded breaks in G1 cells by X-ray radiation was inhibited by knockdown of RAC2. P38 MAPK interaction with RAC2 resulted in a decrease of functional RAC2. Increased phosphorylation of P38 MAPK in G0 cells also increased cellular radioresistance; however, excessive production of ROS caused P38 MAPK dephosphorylation. P38 MAPK, phosphorylated P38 MAPK, and RAC2 regulated in mutual feedback and negative feedback regulatory pathways, resulting in the radioresistance of G0 cells.

By considering both cellular repair effects and indirect effects of radiation, we have generalized the traditional target model, and made it have a linear-quadratic-linear characteristic. To assess the repair capacity-dependent radiosensitivity and relative biological effectiveness (RBE), the generalized target model was used to fit the survival of human normal embryonic lung fibroblast MRC-5 cells in the G0 and G1 phases after various types of radiations. The fitting results indicate that the generalized target model works well in the dose ranges considered. The resulting calculations qualitatively show that the parameter ratio (a/V) in the model could represent the cellular repair capacity. In particular, the significant linear correlations between radiosensitivity/RBE and cellular repair capacity are observed for different slopes of the linear regression curves. These results show that the radiosensitivity and RBE depend on the cellular repair capacity and can be regulated by linear energy transfer. These analyses suggest that the ratio a/V in the generalized target model can also be used for radiation damage assessment in radiotherapy., (© The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

Purpose: To investigate the radiation effects and acute damage in inoperable cervical cancer patients irradiated at different times as well as the underlying mechanisms., Methods: 67 patients were randomized to a morning group (MG, 9:00 - 11:00 AM) and an evening group (EG, 9:00 - 11:00 PM) and both received external beam radiotherapy (RT) (50 Gy in 25 fractions) at different times. Brachytherapy (36 - 42 Gy in 6 - 7 fractions) was also performed to enhance the radiation response twice every week in all patients at the same time. Clinical therapeutic effects and acute toxicities were evaluated after RT. Flow cytometry was analyzed before and after RT., Results: Patients' response to radiation was similar in the two groups. Incidences of overall and high-grade (III - IV) diarrhea in the MG vs. the EG were 75.0% vs. 57.6% and 12.5% vs. 6.1%, respectively. The incidence of severe hematological toxicity in the EG was significantly increased compared to the MG group. Cell apoptosis in the EG was significantly higher at 9:00 - 11:00 PM than that at 9:00 - 11:00 AM after RT. No significant differences were found in Gap Phase 0/Gap Phase 1 (G0/G1), Gap Phase 2/Metaphase Phase (G2/M), and Synthesis Phase (S) phase between different times and groups, nor were expressions of Per1, Per2, and Clock. But expressions of Per1, Per2, and Clock were significantly negative with G2/M phase and positively correlated with cell apoptosis., Conclusion: RT at different time intervals results in similar efficacy. However, RT in the morning reduces severe hematological toxicity. Radiation responses may be associated with circadian genes by influence of cell cycles and apoptosis.
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The DNA damage response (DDR) is a coordinated signaling network that ensures the maintenance of genome stability under DNA damaging stress. In response to DNA lesions, activation of the DDR leads to the establishment of cell cycle checkpoints that delay cell-cycle progression and allow repair of the defects. The tumor suppressor p27Kip1 is a cyclin-CDK inhibitor that plays an important role in regulating quiescence in a variety of tissues. Several studies have suggested that p27Kip1 also plays a role in the maintenance of genomic integrity. Here we demonstrate that p27Kip1 is essential for the establishment of a G1 checkpoint arrest after DNA damage. We also uncovered that ATM phosphorylates p27Kip1 on a previously uncharacterized residue (Ser-140), which leads to its stabilization after induction of DNA double-strand breaks. Inhibition of this stabilization by replacing endogenous p27Kip1 with a Ser-140 phospho-mutant (S140A) significantly sensitized cells to IR treatments. Our findings reveal a novel role for p27Kip1 in the DNA damage response pathway and suggest that part of its tumor suppressing functions relies in its ability to mediate a G1 arrest after the induction of DNA double strand breaks., Competing Interests: The authors have declared that no competing interests exist.

Development of therapeutic resistance is responsible for most prostate cancer (PCa) related mortality. Resistance has been attributed to an acquired or selected cancer stem cell phenotype. Here we report the histone deacetylase inhibitor apicidin (APC) or ER stressor thapsigargin (TG) potentiate paclitaxel (TXL)-induced apoptosis in PCa cells and limit accumulation of cancer stem cells. TXL-induced responses were modulated in the presence of TG with increased accumulation of cells at G1-phase, rearrangement of the cytoskeleton, and changes in cytokine release. Cytoskeletal rearrangement was associated with modulation of the cytoplasmic and mitochondrial unfolded protein response leading to mitochondrial dysfunction and release of proapoptotic proteins from mitochondria. TXL in combination with APC or TG enhanced caspase activation. Importantly, TXL in combination with TG induced caspase activation and apoptosis in X-ray resistant LNCaP cells. Increased release of transforming growth factor-beta (TGF-β) was observed while phosphorylated β-catenin level was suppressed with TXL combination treatments. This was accompanied by a decrease in the CD44(+)CD133(+) cancer stem cell-like population, suggesting treatment affects cancer stem cell properties. Taken together, combination treatment with TXL and either APC or TG induces efficient apoptosis in both proliferating and cancer stem cells, suggesting this therapeutic combination may overcome drug resistance and recurrence in PCa., (Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

DNA double-strand breaks (DSBs) are potentially lethal lesions repaired by two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). Homologous recombination preferentially reunites cognate broken ends. In contrast, non-homologous end-joining could ligate together any two ends, possibly generating dicentric or acentric fragments, leading to inviability. Here, we characterize the yeast NHEJ pathway in populations of pure G1 phase cells, where there is no possibility of repair using a homolog. We show that in G1 yeast cells, NHEJ is a highly effective repair pathway for gamma-ray induced breaks, even when many breaks are present. Pulsed-field gel analysis showed chromosome karyotypes following NHEJ repair of cells from populations with multiple breaks. The number of reciprocal translocations was surprisingly low, perhaps zero, suggesting that NHEJ preferentially re-ligates the "correct" broken ends instead of randomly-chosen ends. Although we do not know the mechanism, the preferential correct ligation is consistent with the idea that broken ends are continuously held together by protein-protein interactions or by larger scale chromatin structure., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Ionizing radiation (IR) exerts deleterious effects on the developing brain, since proliferative neuronal progenitor cells are highly sensitive to IR-induced DNA damage. Assuming a radiation response that is comparable to mammals, the chick embryo would represent a lower vertebrate model system that allows analysis of the mechanisms underlying this sensitivity, thereby contributing to the reduction, refinement and replacement of animal experiments. Thus, this study aimed to elucidate the radiation response of the embryonic chick retina in three selected embryonic stages. Our studies reveal a lack in the radiation-induced activation of a G1/S checkpoint, but rapid abrogation of G2/M progression after IR in retinal progenitors throughout development. Unlike cell cycle control, radiation-induced apoptosis (RIA) showed strong variations between its extent, dose dependency and temporal occurrence. Whereas the general sensitivity towards RIA declined with ongoing differentiation, its dose dependency constantly increased with age. For all embryonic stages RIA occurred during comparable periods after irradiation, but in older animals its maximum shifted towards earlier post-irradiation time points. In summary, our results are in good agreement with data from the developing rodent retina, strengthening the suitability of the chick embryo for the analysis of the radiation response in the developing central nervous system.

Low-dose hyper-radiosensitivity (HRS) has been reported in normal human lymphoblastoid cell lines for exposures at ≤ 20 cGy, but the cytogenetic effects of oxygen (O2 ) levels in tissue culture medium on HRS have not been evaluated. We asked whether HRS was lost in G2-irradiated cells grown in atmospheres of 2.5% or 5% O2 , compared to responses by cells cultured in ambient O2 (21%). The results indicate a loss of HRS when cells are cultured and irradiated either in 2.5% or 5% O2 . We then evaluated whether low O2 levels either before or after exposure were responsible for the loss of HRS. For cells irradiated in 5% O2 , subsequent immediate re-oxygenation to ambient O2 levels restored the HRS effect, while cells cultured and irradiated at ambient O2 levels and then transferred to 5% O2 exhibited little or no HRS, indicating that ambient O2 levels after, but not before, radiation substantially affect the amounts of cytogenetic damage. HRS was not observed when cells were irradiated in G1. At doses of 40-400 cGy there was significantly less cytogenetic damage when cells were recovering from radiation at low O2 levels than at ambient O2 levels. Here we provide the first cytogenetic evidence for the loss of HRS at low O2 levels in G2-irradiated cells; these results suggest that at low O2 levels for all doses evaluated there is either less damage to DNA, perhaps because of lower amounts of reactive oxygen species, or that DNA damage repair pathways are activated more efficiently., (© 2015 Wiley Periodicals, Inc.)

Using an asynchronously growing cell population, we investigated how X-irradiation at different stages of the cell cycle influences individual cell-based kinetics. To visualize the cell-cycle phase, we employed the fluorescent ubiquitination-based cell cycle indicator (Fucci). After 5 Gy irradiation, HeLa cells no longer entered M phase in an order determined by their previous stage of the cell cycle, primarily because green phase (S and G2) was less prolonged in cells irradiated during the red phase (G1) than in those irradiated during the green phase. Furthermore, prolongation of the green phase in cells irradiated during the red phase gradually increased as the irradiation timing approached late G1 phase. The results revealed that endoreduplication rarely occurs in this cell line under the conditions we studied. We next established a method for classifying the green phase into early S, mid S, late S, and G2 phases at the time of irradiation, and then attempted to estimate the duration of G2 arrest based on certain assumptions. The value was the largest when cells were irradiated in mid or late S phase and the smallest when they were irradiated in G1 phase. In this study, by closely following individual cells irradiated at different cell-cycle phases, we revealed for the first time the unique cell-cycle kinetics in HeLa cells that follow irradiation.

Purpose: To investigate the dependence of the bystander cell-killing effect on radiation dose and quality, and to elucidate related molecular mechanisms., Materials and Methods: Normal human fibroblast WI-38 cells were irradiated with 0.125 - 2 Gy of γ-rays or carbon ions and were co-cultured with non-irradiated cells. Survival rates of bystander cells were investigated using the colony formation assays, and nitrite concentrations in the medium were measured using the modified Saltzman method., Results: Survival rates of bystander cells decreased with doses of γ-rays and carbon ions of ≤ 0.5 Gy. Treatment of the specific nitric oxide (NO) radical scavenger prevented reductions in survival rates of bystander cells. Moreover, nitrite concentrations increased with doses of less than 0.25 Gy (γ-rays) and 1 Gy (carbon ions). The dose responses of increased nitrite concentrations as well as survival reduction were similar between γ-rays and carbon ions. In addition, negative relationships were observed between survival rates and nitrite concentrations., Conclusion: The bystander cell-killing effect mediated by NO radicals in normal human fibroblasts depends on irradiation doses of up to 0.5 Gy, but not on radiation quality. NO radical production appears to be an important determinant of γ-ray- and carbon-ion-induced bystander effects.

The present study was aimed to investigate whether combination of molecular targeting therapy, a dual PI3K/mTOR inhibitor (BEZ235), with radiation can enhance the radiosensitivity of colorectal cancer cells (CRC). K-RAS mutant CRC cells (HCT 116 and SW 620) and wild type CRC cells (HT 29) were irradiated with different dose of radiation (0-6 Gy). The synergistic effects of combining radiation with different concentration of BEZ235 (0-10 nM) pretreatment were demonstrated by cell survival assay. When comparing with radiation alone and BEZ235 alone, the combination of BEZ235 pretreatment and radiation resulted in an increased percentage of sub-G1 phase cells, and an increased number of γ-H2AX/cell (DNA double strand breaks). Radiation up-regulated AKT/mTOR signaling pathway, including the activation of phospho (p)-AKT, p-mTOR, p-eIF4E, and p-rpS6; and this activated AKT/mTOR signaling pathway was attenuated by BEZ235 pretreatment. In addition, BEZ235 blocked double strand break repair induced by radiation through attenuating the activation of ATM and DNA-PKcs and sensitized CRC cells to radiation. In vivo model, the tumor size and the expression pattern of p-mTOR, p-eIF4E, and p-rpS6 were significantly decreased in combined group than radiation alone or BEZ235 alone. Our findings indicate that the administration of BEZ235 before radiation enhances the radiotherapeutic effect of CRC cells both in vitro and in vivo., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The purpose of this work is to test the hypothesis that kinetics of double strand breaks (DSB) repair is governed by complexity of DSB. To test the hypothesis we used our recent published mechanistic mathematical model of DSB repair for DSB induced by selected protons, deuterons, and helium ions of different energies representing radiations of different qualities. In light of recent advances in experimental and computational techniques, the most appropriate method to study cellular responses in radiation therapy, and exposures to low doses of ionizing radiations is using mechanistic approaches. To this end, we proposed a 'bottom-up' approach to study cellular response that starts with the DNA damage. Monte Carlo track structure method was employed to simulate initial damage induced in the genomic DNA by direct and indirect effects. Among the different types of DNA damage, DSB are known to be induced in simple and complex forms. The DSB repair model in G1 and early S phases of the cell cycle was employed to calculate the repair kinetics. The model considers the repair of simple and complex DSB, and the DSB produced in the heterochromatin. The inverse sampling method was used to calculate the repair kinetics for each individual DSB. The overall repair kinetics for 500 DSB induced by single tracks of the radiation under test were compared with experimental results. The results show that the model is capable of predicting the repair kinetics for the DSB induced by radiations of different qualities within an accepted range of uncertainty., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We previously reported that an optimal dose of lipopolysaccharide (LPS) markedly extends the lifespan of murine primary-cultured microglia by suppressing cell death pathways. In this study, we investigated the effects of LPS pretreatment on UV light-induced apoptosis of cells from the microglial cell line BV-2. More than half of BV-2 cells were apoptotic, and procaspase-3 was cleaved into its active form at 3 h of UV irradiation. In contrast, in BV-2 cells treated with LPS for 24 h, UV irradiation caused neither apoptosis nor procaspase-3 cleavage. LPS treatment arrested the cell cycle in G1 phase and upregulated cyclin-dependent kinase inhibitor p21(Waf1/Cip1) and growth arrest and DNA damage-inducible (GADD) 45α in BV-2 cells. When p21(Waf1/Cip1) and GADD45α were knocked down by small interfering RNA, procaspase-3 was cleaved into its active form to induce apoptosis. Our findings suggest that LPS inhibits UV-induced apoptosis in BV-2 cells through arrest of the cell cycle in G1 phase by upregulation of p21(Waf1/Cip1) and GADD45α. Excessive activation of microglia may play a critical role in the exacerbation of neurodegeneration, therefore, normalizing the precise regulation of apoptosis may be a new strategy to prevent the deterioration caused by neurodegenerative disorders.

Terahertz (THz) radiation was proposed recently for use in various applications, including medical imaging and security scanners. However, there are concerns regarding the possible biological effects of non-ionising electromagnetic radiation in the THz range on cells. Human embryonic stem cells (hESCs) are extremely sensitive to environmental stimuli, and we therefore utilised this cell model to investigate the non-thermal effects of THz irradiation. We studied DNA damage and transcriptome responses in hESCs exposed to narrow-band THz radiation (2.3 THz) under strict temperature control. The transcription of approximately 1% of genes was subtly increased following THz irradiation. Functional annotation enrichment analysis of differentially expressed genes revealed 15 functional classes, which were mostly related to mitochondria. Terahertz irradiation did not induce the formation of γH2AX foci or structural chromosomal aberrations in hESCs. We did not observe any effect on the mitotic index or morphology of the hESCs following THz exposure.

Exposure of cells to ionizing radiation (IR) induces, not only, activation of multiple signaling pathways that play critical roles in cell fate determination, but also alteration of molecular pathways involved in cell death or survival. Recently, DNA methylation has been established as a critical epigenetic process involved in the regulation of gene expression in cancer cells, suggesting that DNA methylation inhibition may be an effective cancer treatment strategy. Because alterations of gene expression by DNA methylation have been considered to influence radioresponsiveness, we investigated the effect of a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), on radiosensitivity. In addition, we investigated the underlying cellular mechanisms of combination treatments of ionizing irradiation (IR) and 5-aza-dC in human colon cancer cells. Colon cancer cell lines were initially tested for radiation sensitivity by IR in vitro and were treated with two different doses of 5-aza-dC. Survival of these cell lines was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and clonogenic assays. The effects of 5-aza-dC along with irradiation on cell growth, cell cycle distribution, apoptosis, and apoptosis-related gene expression were examined. Combination irradiation treatment with 5-aza-dC significantly decreased growth activity compared with irradiation treatment alone or with 5-aza-dC treatment alone. The percentage of HCT116 cells in the sub-G1 phase and their apoptotic rate was increased when cells were treated with irradiation in combination with 5-aza-dC compared with either treatment alone. These observations were strongly supported by increased caspase activity, increased comet tails using comet assays, and increased protein levels of apoptosis-associated molecules (caspase 3/9, cleaved PARP). Our data demonstrated that 5-aza-dC enhanced radiosensitivity in colon cancer cells, and the combination effects of 5-aza-dC with radiation showed greater cellular effects than that of single treatment, suggesting that the combination of 5-aza-dC and radiation has the potential to become a clinical strategy for the treatment of cancer.

The basic machinery that detects DNA damage is the same throughout the cell cycle. Here, we show, in contrast, that reversal of DNA damage responses (DDRs) and recovery are fundamentally different in G1 and G2 phases of the cell cycle. We find that distinct phosphatases are required to counteract the checkpoint response in G1 vs. G2. Whereas WT p53-induced phosphatase 1 (Wip1) promotes recovery in G2-arrested cells by antagonizing p53, it is dispensable for recovery from a G1 arrest. Instead, we identify phosphoprotein phosphatase 4 catalytic subunit (PP4) to be specifically required for cell cycle restart after DNA damage in G1. PP4 dephosphorylates Krüppel-associated box domain-associated protein 1-S473 to repress p53-dependent transcriptional activation of p21 when the DDR is silenced. Taken together, our results show that PP4 and Wip1 are differentially required to counteract the p53-dependent cell cycle arrest in G1 and G2, by antagonizing early or late p53-mediated responses, respectively.

Objective: To investigate the effect of RING finger protein 2 (RNF2) knockdown on the biological characteristics and radiosensitivity in glioma U87 cells., Methods: Plasmids containing shRNA targeting RNF2 were transfected into U87 cells. Real-time quantitative PCR (qRT-PCR) and Western blotting were respectively applied to detect the mRNA and protein level of RNF2. MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were measured by flow cytometry combined with annexin V-FITC/PI staining in the control and RNF2 knockdown cells. Apoptosis was also detected after X-ray radiation., Results: Both shRNAs efficiently inhibited RNF2 expression in U87 cells. Cell proliferation was obviously depressed in RNF2 knockdown cells. The percentage of cells decreased in S phase (shRNA-NC: 27.31 ± 1.35; shRNF2-1: 16.72 ± 2.90; shRNF2-3: 10.35 ± 1.33) and increased in G1 phase (shRNA-NC: 56.13 ± 1.80; shRNF2-1: 76.32 ± 3.11; shRNF2-3: 80.45 ± 2.83). More cell apoptosis was observed in RNF2 knockdown cells. After X ray radiation, the apoptosis rate was significantly raised in RNF2 knockdown cells (shRNA-NC: 20.88 ± 0.64; shRNF2-1: 39.69 ± 0.57; shRNF2-3: 47.82 ± 0.45)., Conclusion: Knockdown of RNF2 can inhibit cell proliferation, induce cell cycle arrest and promote apoptosis in U87 cells. RNF2 knockdown can obviously increase the sensitivity of U87 cells to X ray radiation.

Background and Purpose: Novel radiotherapy techniques increasingly use very large dose fractions. It has been argued that the biological effect of large dose fractions may differ from that of conventional fraction sizes. The purpose was to study the biological effect of large single doses., Material and Methods: Clonogenic cell survival of MCF7 and MDA-MB-231 cells was determined after direct X-ray irradiation, irradiation of feeder cells, or transfer of conditioned medium (CM). Cell-cycle distributions and the apoptotic sub-G1 fraction were measured by flow cytometry. Cytokines in CM were quantified by a cytokine antibody array. γH2AX foci were detected by immunofluorescence microscopy., Results: The surviving fraction of MCF7 cells irradiated in vitro with 12 Gy showed an 8.5-fold decrease (95% c.i.: 4.4-16.3; P<0.0001) when the density of irradiated cells was increased from 10 to 50×10(3) cells per flask. Part of this effect was due to a dose-dependent transferrable factor as shown in CM experiments in the dose range 5-15 Gy. While no effect on apoptosis and cell cycle distribution was observed, and no differentially expressed cytokine could be identified, the transferable factor induced prolonged expression of γH2AX DNA repair foci at 1-12 h., Conclusions: A dose-dependent non-targeted effect on clonogenic cell survival was found in the dose range 5-15 Gy. The dependence of SF on cell numbers at high doses would represent a "cohort effect" in vivo. These results support the hypothesis that non-targeted effects may contribute to the efficacy of very large dose fractions in radiotherapy.

The kinase ATR is activated by RPA-coated single-stranded DNA generated at aberrant replicative structures and resected double strand breaks. While many hundred candidate ATR substrates have been identified, the essential role of ATR in the replicative stress response has impeded the study of ATR kinase-dependent signalling. Using recently developed selective drugs, we show that ATR inhibition has a significantly more potent effect than ATM inhibition on ionizing radiation (IR)-mediated cell killing. Transient ATR inhibition for a short interval after IR has long-term consequences that include an accumulation of RPA foci and a total abrogation of Chk1 S345 phosphorylation. We show that ATR kinase activity in G1 phase cells is important for survival after IR and that ATR colocalizes with RPA in the absence of detectable RPA S4/8 phosphorylation. Our data reveal that, unexpectedly, ATR kinase inhibitors may be more potent cellular radiosensitizers than ATM kinase inhibitors, and that this is associated with a novel role for ATR in G1 phase cells.

Ionizing radiation (IR) induces genotoxic stress that triggers adaptive cellular responses, such as activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade. Pluripotent cells are the most important population affected by IR because they are required for cellular replenishment. Despite the clear danger to large population centers, we still lack safe and effective therapies to abrogate the life-threatening effects of any accidental or intentional IR exposure. Therefore, we computationally analyzed the chemical structural similarity of previously published small molecules that, when given after IR, mitigate cell death and found a chemical cluster that was populated with PI3K inhibitors. Subsequently, we evaluated structurally diverse PI3K inhibitors. It is remarkable that 9 of 14 PI3K inhibitors mitigated γIR-induced death in pluripotent NCCIT cells as measured by caspase 3/7 activation. A single intraperitoneal dose of LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], administered to mice at 4 or 24 hours, or PX-867 [(4S,4aR,5R,6aS,9aR,Z)-11-hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-2,7,10-trioxo-1-(pyrrolidin-1-ylmethylene)-1,2,4,4a,5,6,6a,7,8,9,9a,10-dodecahydroindeno[4,5-H]isochromen-5-yl acetate (CID24798773)], administered 4 hours after a lethal dose of γIR, statistically significantly (P < 0.02) enhanced in vivo survival. Because cell cycle checkpoints are important regulators of cell survival after IR, we examined cell cycle distribution in NCCIT cells after γIR and PI3K inhibitor treatment. LY294002 and PX-867 treatment of nonirradiated cells produced a marked decrease in S phase cells with a concomitant increase in the G1 population. In irradiated cells, LY294002 and PX-867 treatment also decreased S phase and increased the G1 and G2 populations. Treatment with LY294002 or PX-867 decreased γIR-induced DNA damage as measured by γH2AX, suggesting reduced DNA damage. These results indicate pharmacologic inhibition of PI3K after IR abrogated cell death.

Many proteins exist and function as homodimers. Understanding the detailed mechanism driving the homodimerization is important and will impact future studies targeting the "undruggable" oncogenic protein dimers. In this study, we used 14-3-3σ as a model homodimeric protein and performed a systematic investigation of the potential roles of amino acid residues in the interface for homodimerization. Unlike other members of the conserved 14-3-3 protein family, 14-3-3σ prefers to form a homodimer with two subareas in the dimeric interface that has 180° symmetry. We found that both subareas of the dimeric interface are required to maintain full dimerization activity. Although the interfacial hydrophobic core residues Leu(12) and Tyr(84) play important roles in 14-3-3σ dimerization, the non-core residue Phe(25) appears to be more important in controlling 14-3-3σ dimerization activity. Interestingly, a similar non-core residue (Val(81)) is less important than Phe(25) in contributing to 14-3-3σ dimerization. Furthermore, dissociating dimeric 14-3-3σ into monomers by mutating the Leu(12), Phe(25), or Tyr(84) dimerization residue individually diminished the function of 14-3-3σ in resisting drug-induced apoptosis and in arresting cells at G2/M phase in response to DNA-damaging treatment. Thus, dimerization appears to be required for the function of 14-3-3σ.

DNA damage induced by ultraviolet (UV) radiation can be removed by nucleotide excision repair through two sub-pathways, one general (GGR) and the other specific for transcribed DNA (TCR), and the processing of unrepaired lesions trigger signals that may lead to cell death. These signals involve the tumor suppressor p53 protein, a central regulator of cell responses to DNA damage, and the E3 ubiquitin ligase Mdm2, that forms a feedback regulatory loop with p53. The involvement of cell cycle and transcription on the signaling to apoptosis was investigated in UVB-irradiated synchronized, DNA repair proficient, CS-B (TCR-deficient) and XP-C (GGR-deficient) primary human fibroblasts. Cells were irradiated in the G1 phase of the cell cycle, with two doses with equivalent levels of apoptosis (low and high), defined for each cell line. In the three cell lines, the low doses of UVB caused only a transient delay in progression to the S phase, whereas the high doses induced permanent cell cycle arrest. However, while accumulation of Mdm2 correlated well with the recovery from transcription inhibition at the low doses for normal and CS-B fibroblasts, for XP-C cells this protein was shown to be accumulated even at UVB doses that induced high levels of apoptosis. Thus, UVB-induced accumulation of Mdm2 is critical for counteracting p53 activation and apoptosis avoidance, but its effect is limited due to transcription inhibition. However, in the case of XP-C cells, an excess of unrepaired DNA damage would be sufficient to block S phase progression, which would signal to apoptosis, independent of Mdm2 accumulation. The data clearly discriminate DNA damage signals that lead to cell death, depending on the presence of UVB-induced DNA damage in replicating or transcribing regions.

The paper presents a model of double strand breaks (DSB) repair in G1 and early S phases of the cell cycle. The model is based on a plethora of published information on biochemical modification of DSB induced by ionizing radiation. So far, three main DSB repair pathways have been identified, including nonhomologous end-joining (NHEJ), homologous recombination (HR), and microhomology-mediated end-joining (MMEJ). During G1 and early S phases of the cell cycle, NHEJ and MMEJ repair pathways are activated dependent on the type of double strand breaks. Simple DSB are a substrate for NHEJ, while complex DSB and DSB in heterochromatin require further end processing. Repair of all DSB start with NHEJ presynaptic processes, and depending on the type of DSB pursue simple ligation, further end processing prior to ligation, or resection. Using law of mass action the model is translated into a mathematical formalism. The solution of the formalism provides the step by step and overall repair kinetics. The overall repair kinetics are compared with the published experimental measurements. Our calculations are in agreement with the experimental results and show that the complex types of DSBs are repaired with slow repair kinetics. The G1 and early S phase model could be employed to predict the kinetics of DSB repair for damage induced by high LET radiation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Purpose: Radiation therapy for invasive bladder cancer allows for organ preservation but toxicity and local control remain problematic. As such, improving efficacy of treatment requires radiosensitization of tumor cells. The aim of study is to investigate if the mammalian Target of Rapamycin (mTOR), a downstream kinase of the phosphatidylinositol 3-kinase (PI3K)/AKT survival pathway, may be a target for radiation sensitization., Experimental Design: Clonogenic assays were performed using 6 bladder cancer cell lines (UM-UC3, UM-UC5, UM-UC6, KU7, 253J-BV, and 253-JP) in order to examine the effects of ionizing radiation (IR) alone and in combination with RAD001, an mTOR inhibitor. Cell cycle analysis was performed using flow cytometry. In vivo, athymic mice were subcutaneously injected with 2 bladder cancer cell lines. Treatment response with RAD001 (1.5 mg/kg, daily), fractionated IR (total 9Gy = 3Gy×3), and combination of RAD001 and IR was followed over 4 weeks. Tumor weight was measured at experimental endpoint., Results: Clonogenic assays revealed that in all bladder cell lines tested, an additive effect was observed in the combined treatment when compared to either treatment alone. Our data indicates that this effect is due to arrest in both G1 and G2 phases of cell cycle when treatments are combined. Furthermore, our data show that this arrest is primarily regulated by changes in levels of cyclin D1, p27 and p21 following treatments. In vivo, a significant decrease in tumor weight was observed in the combined treatment compared to either treatment alone or control., Conclusions: Altering cell cycle by inhibiting the mTOR signaling pathway in combination with radiation have favorable outcomes and is a promising therapeutic modality for bladder cancer.

Diploid cells undergoing senescence and mitotic slippage have been reported in the literature. However, the mechanisms triggering senescence in long-term G2-arrested cells are currently unclear. Previously, we reported that the cell cycle of the human uveal melanoma cell line, 92-1, is suspended for up to 6 d upon exposure to 10 Gy ionizing radiation (IR), followed by senescence. In the current study, we initially distinguished senescence in long-term blocked 92-1 cells from mitotic slippage by confirming the blockage of cells in the G2 phase. We subsequently showed that the genes essential for G2-M transition are prematurely downregulated at both the transcriptional and translational levels. Furthermore, levels of the G1-specific markers, Cyclin D1 and Caveolin-1, were distinctly increased, while S/G2-specific markers, Cyclin B1 and Aurora A, were significantly downregulated. These findings collectively imply that long-term G2-arrested cells undergo senescence via G2 slippage. To our knowledge, this is the first study to report that the cellular process of G2 slippage is the mechanism responsible for senescence of cells under long-term G2 arrest.

Cancer patients often suffer from local tumor recurrence after radiation therapy. Some intracellular and extracellular factors, such as activity of hypoxia-inducible factor 1 (HIF-1), cell cycle status and oxygen availability, have been suggested to affect DNA damage responses and eventual radioresistant characteristics of cancer cells. But when, where, and how these factors affect one another and induce cellular radioresistance is largely unknown. Here, we analyzed mechanistic and spatio-temporal relationships among them in highly heterogeneous tumor microenvironments. Experiments in vitro demonstrated that a decrease in the glucose concentration reduced the transcriptional activity of HIF-1 and expression of a downstream gene for the cell cycle regulator p27(Kip1) even under hypoxic conditions. Then, the proportion of cells in the radioresistant S phase increased, whereas that in the radiosensitive G1 phase decreased, significantly. Immunohistochemical analyses showed that cancer cells in perinecrotic hypoxic regions, which should be under low-glucose conditions, expressed little HIF-1α, and therefore, were mainly in S phase and less damaged by radiation treatment. Continuous administration of glucagon, which increases the blood glucose concentration and so improves glucose availability in perinecrotic hypoxic regions, induced HIF-1α expression and increased radiation-induced DNA damage. Taken all together, these results indicate that cancer cells in perinecrotic regions, which would be under low-glucose and hypoxic conditions, obtain radioresistance by decreasing the level of both HIF-1 activity and p27(Kip1) expression, and adjusting their cell cycle to the radioresistant S phase.

Given the promising control of bladder cancer achieved by combined chemotherapy/radiotherapy with selective transurethral resection, obstacles remain to the treatment of unresectable bladder cancer. The aim of this study was to determine whether targeting epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) can radiosensitise a murine bladder tumour (MBT-2) cell line. Cell survival, expression of signal proteins and cell cycle changes in MBT-2 cells treated in vitro and in vivo with afatinib, an irreversible EGFR/HER2 inhibitor, plus radiotherapy were investigated by colony formation assay, Western blot assay and flow cytometry, respectively. Ectopic xenografts were established by subcutaneous injection of MBT-2 cells in C3H/HeN mice. Mice were randomised into 4 groups to receive afatinib (10mg/kg/day on day 1-7) and/or radiotherapy (15Gy on day 4). Positron emission tomography (PET) on day 8 was used to evaluate the early treatment response. Afatinib (200-1000nM) increased cell killing by radiation (0-10Gy). Pre-treatment of irradiated cells with afatinib inhibited radiation-activated HER2 and EGFR phosphorylation. As compared to either treatment alone, the combination increased the level of the cleavage form of poly (ADP-ribose) polymerase, the expression of phospho-γH2AX and the percentage of cells in subG1 phase (indicating enhanced induction of apoptosis), and decreased tumour metabolism and inhibited tumour growth by 64%. Afatinib has therapeutic value as a radiosensitiser of murine bladder cancer cells. The synergism between afatinib and radiation likely enhances DNA damage, leading to increased cell apoptosis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Genomic instability stemming from dysregulation of cell cycle checkpoints and DNA damage response (DDR) is a common feature of many cancers. The cancer adult T cell leukemia (ATL) can occur in individuals infected with human T cell leukemia virus type 1 (HTLV-1), and ATL cells contain extensive chromosomal abnormalities, suggesting that they have defects in the recognition or repair of DNA damage. Since Tax is the transforming protein encoded by HTLV-1, we asked whether Tax can affect cell cycle checkpoints and the DDR. Using a combination of flow cytometry and DNA repair assays we showed that Tax-expressing cells exit G(1) phase and initiate DNA replication prematurely following damage. Reduced phosphorylation of H2AX (γH2AX) and RPA2, phosphoproteins that are essential to properly initiate the DDR, was also observed in Tax-expressing cells. To determine the cause of decreased DDR protein phosphorylation in Tax-expressing cells, we examined the cellular phosphatase, WIP1, which is known to dephosphorylate γH2AX. We found that Tax can interact with Wip1 in vivo and in vitro, and that Tax-expressing cells display elevated levels of Wip1 mRNA. In vitro phosphatase assays showed that Tax can enhance Wip1 activity on a γH2AX peptide target by 2-fold. Thus, loss of γH2AX in vivo could be due, in part, to increased expression and activity of WIP1 in the presence of Tax. siRNA knockdown of WIP1 in Tax-expressing cells rescued γH2AX in response to damage, confirming the role of WIP1 in the DDR. These studies demonstrate that Tax can disengage the G(1)/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G(1) exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome.

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G(0) and G(1) phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.

Purpose: To investigate the injury of murine mesenchymal stem cells (mMSC) exposed to 4 Gy X-radiation and the role of canonical and non-canonical wingless-type (Wnt) signaling in the radiation injury., Materials and Methods: C3H10T1/2 cells were submitted to 4 Gy X-radiation. At different time points after radiation, Hoechst33258 staining and Annexin V-fluorescein isothiocyanate (FITC) flow cytometry analysis were performed to assess cellular apoptosis. Senescence-associated β-galactosidase (SA-β-gal) staining was performed to analyze cellular senescence. Cell cycle was measured by flow cytometry. P53, p21, Wnt3a, Wnt5a, sonic hedgehog (Shh) mRNA was detected by Real time polymerase chain reaction (PCR) and Wnt5a protein was determined by Western blot., Results: A time-dependent cellular apoptosis was observed with a peak level 12 hours after radiation. Cellular senescence was detected 72 h after radiation. A remarkable up-regulation of Wnt5a mRNA expression (∼ 269-fold) and protein expression was seen 72 h after radiation., Conclusions: The effect of 4 Gy X-radiation to mMSC was time-dependent in the form of cellular apoptosis in the early period and cellular senescence in the late period. Non-canonical Wnt signaling may be involved in mMSC senescence induced by 4 Gy X-radiation.

A series of meso-substituted tetra-cationic porphyrins, which have methyl and octyl substituents, was studied in order to understand the effect of zinc chelation and photosensitizer subcellular localization in the mechanism of cell death. Zinc chelation does not change the photophysical properties of the photosensitizers (all molecules studied are type II photosensitizers) but affects considerably the interaction of the porphyrins with membranes, reducing mitochondrial accumulation. The total amount of intracellular reactive species induced by treating cells with photosensitizer and light is similar for zinc-chelated and free-base porphyrins that have the same alkyl substituent. Zinc-chelated porphyrins, which are poorly accumulated in mitochondria, show higher efficiency of cell death with features of apoptosis (higher MTT response compared with trypan blue staining, specific acridine orange/ethidium bromide staining, loss of mitochondrial transmembrane potential, stronger cytochrome c release and larger sub-G1 cell population), whereas nonchelated porphyrins, which are considerably more concentrated in mitochondria, triggered mainly necrotic cell death. We hypothesized that zinc-chelation protects the photoinduced properties of the porphyrins in the mitochondrial environment., (© 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.)

Purpose: Mutations in the epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/Akt signaling transduction pathway are common in cancer. This pathway is imperative to the radiosensitivity of cancer cells. We aimed to investigate the radiosensitizing effects of the simultaneous inhibition of EGFR and PI3K in breast cancer cells., Methods and Materials: MCF-7 cell lines with low expression of EGFR and wild-type PTEN and MDA-MB-468 cell lines with high expression of EGFR and mutant PTEN were used. The radiosensitizing effects by the inhibition of EGFR with AG1478 and/or PI3K with Ly294002 were determined by colony formation assay, Western blot was used to investigate the effects on downstream signaling. Flow cytometry was used for apoptosis and cell cycle analysis. Mice-bearing xenografts of MDA-MB-468 breast cancer cells were also used to observe the radiosensitizing effect., Results: Simultaneous inhibition of EGFR and PI3K greatly enhanced radiosensitizing effect in MDA-MB-468 in terms of apoptosis and mitotic death, either inhibition of EGFR or PI3K alone could enhance radiosensitivity with a dose-modifying factor (DMF(SF2)) of 1.311 and 1.437, radiosensitizing effect was further enhanced by simultaneous inhibition of EGFR and PI3K with a DMF(SF2) at 2.698. DNA flow cytometric analysis indicated that dual inhibition combined with irradiation significantly induced G0/G1 phase arrest in MDA-MB-468 cells. The expression of phosphor-Akt and phosphor-Erk1/2 (induced by irradiation and PI3K inhibitor) were fully attenuated by simultaneous treatment with both inhibitors in combination with irradiation. In addition, dual inhibition combined with irradiation induced dramatic tumor growth delay in MDA-MB-468 xenografts., Conclusions: Our study indicated that simultaneous inhibition of EGFR and PI3K could further sensitize the cancer cells to irradiation compared to the single inhibitor with irradiation in vitro and in vivo. The approach may have important therapeutic implication in the treatment of a subset of breast cancer patients with high expression of EGFR and deficient function of PTEN., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Transforming growth factor-β-activated kinase 1 (TAK1) appears to play a role in inhibiting apoptotic death in response to multiple stresses. To assess the role of TAK1 in X-ray induced apoptosis and cell death, we irradiated parental and siRNA-TAK1-knockdown HeLa cells. Changes in gene expression levels with and without TAK1-knockdown were also examined after irradiation to elucidate the molecular mechanisms involved. After X-ray irradiation, cell death estimated by the colony formation assay increased in the TAK1-knockdown cells. Apoptosis induction, determined by caspase-3 cleavage, suggested that the increased radiosensitivity of the TAK1-knockdown cells could be partially explained by the induction of apoptosis. However, cell cycle analysis revealed that the percentage of irradiated cells in the G(2)/M-phase decreased, and those in the S- and SubG(1)-phases increased due to TAK1 depletion, suggesting that the loss of cell cycle checkpoint regulation may also be involved in the observed increased radiosensitivity. Interestingly, significant differences in the induction of NF-κB, p38 MAPK and ERK phosphorylation, the major downstream molecules of TAK1, were not observed in TAK1 knockdown cells compared to their parental control cells after irradiation. Instead, global gene expression analysis revealed differentially expressed genes after irradiation that bioinformatics analysis suggested are associated with cell cycle regulatory networks. In particular, CDKN1A (coding p21(WAF1)), which plays a central role in the identified network, was up-regulated in control cells but not in TAK1 knockdown cells after X-ray irradiation. Si-RNA knockdown of p21 decreased the percentage of cells in the G(2)/M phase and increased the percentage of cells in the S- and SubG(1)-phases after X-ray irradiation in a similar manner as TAK-1 knockdown. Taken together, these findings suggest that the role of TAK1 in cell death, cell cycle regulation and apoptosis after X irradiation is independent of NF-κB, p38 MAPK, and ERK phosphorylation, and dependent, in part, on p21 induction.

UVR is a major environmental risk factor for the development of melanoma. Here we describe a coupled DNA-damage tolerance (DDT) mechanism and G2-phase cell cycle checkpoint induced in response to suberythemal doses of UVR that is commonly defective in melanomas. This coupled response is triggered by a small number of UVR-induced DNA lesions incurred during G1 phase that are not repaired by nucleotide excision repair (NER). These lesions are detected during S phase, but rather than stalling replication, they trigger the DDT-dependent formation of single-stranded DNA (ssDNA) gaps. The ssDNA attracts replication protein A (RPA), which initiates ATR-Chk1 (ataxia telangiectasia and Rad3-related/checkpoint kinase 1) G2-phase checkpoint signaling, and colocalizes with components of the RAD18 and RAD51 postreplication repair pathways. We demonstrate that depletion of RAD18 delays both the resolution of RPA foci and exit from the G2-phase arrest, indicating the involvement of RAD18-dependent postreplication repair in ssDNA gap repair during G2 phase. Moreover, the presence of RAD51 and BRCA1 suggests that an error-free mechanism may also contribute to repair. Loss of the UVR-induced G2-phase checkpoint results in increased UVR signature mutations after exposure to suberythemal UVR. We propose that defects in the UVR-induced G2-phase checkpoint and repair mechanism are likely to contribute to melanoma development.

Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays., Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays., Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO(2)-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ß-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function., Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The translationally controlled tumor protein (TCTP) is essential for survival by mechanisms that as yet are incompletely defined. Here we describe an important role of TCTP in response to DNA damage. Upon exposure of normal human cells to low-dose γ rays, the TCTP protein level was greatly increased, with a significant enrichment in nuclei. TCTP up-regulation occurred in a manner dependent on ataxia-telangiectasia mutated (ATM) kinase and the DNA-dependent protein kinase and was associated with protective effects against DNA damage. In chromatin of irradiated cells, coimmunoprecipitation experiments showed that TCTP forms a complex with ATM and γH2A.X, in agreement with its distinct localization with the foci of the DNA damage-marker proteins γH2A.X, 53BP1, and P-ATM. In cells lacking TCTP, repair of chromosomal damage induced by γ rays was compromised significantly. TCTP also was shown to interact with p53 and the DNA-binding subunits, Ku70 and Ku80, of DNA-dependent protein kinase. TCTP knockdown led to decreased levels of Ku70 and Ku80 in nuclei of irradiated cells and attenuated their DNA-binding activity. It also attenuated the radiation-induced G(1) delay but prolonged the G(2) delay. TCTP therefore may play a critical role in maintaining genomic integrity in response to DNA-damaging agents.

X-ray induced formation of micronuclei is generally thought to result from DNA double-strand breaks (DSBs). However, DNA DSBs inhibit the cell cycle progression that is required for micronucleus formation. In order to reconcile this apparent discrepancy, we investigated whether DNA DSBs induced during the G1 phase could lead to micronucleus formation. We irradiated human embryonic (HE17) cells that had been treated with a radical scavenger, either DMSO or ascorbic acid (AsA), and determined the level of suppression of DNA DSBs or micronuclei. When DNA DSBs were evaluated using 53BP1 foci, treatment with 5 mM AsA did not inhibit the numbers of foci at various intervals after X-ray irradiation; however, treatment with 5 mM or 256 mM DMSO did have a significant inhibitory effect. By contrast, an assay of micronucleus numbers showed that treatment with 5 mM or 256 mM DMSO before X-ray irradiation resulted in almost no inhibition of micronucleus formation, but treatment with 5 mM AsA did have a significant inhibitory effect. These results clearly showed that AsA could suppress micronucleus formation, although it was not effective for suppression of DNA DSBs. Therefore, we conclude that DNA DSBs induced in the G1 phase do not directly lead to micronucleus formation.

Photodynamic therapy (PDT) dosimetry is complex as many factors are involved and varied interdependently. Monitoring the biological consequence of PDT such as cell death is the most direct approach to assess treatment efficacy. In this study, we performed 5-aminolevlinic acid (ALA)-PDT in vitro to induce different cell death modes (i.e., slight cell cytotoxicity, apoptosis, and necrosis) by a fixed fluence rate of 10 mW/cm(2) and varied fluences (1, 2, and 6 J/cm(2)). Time course measurements of cell viability, caspase-3 activity, and DNA fragmentation were conducted to determine the mode of cell death. We demonstrated that NADH fluorescence lifetime together with NADH fluorescence intensity permit us to detect apoptosis and differentiate it from necrosis. This feature will be unique in the use of optimizing apoptosis-favored treatments such as metronomic PDT., (© 2011 Optical Society of America)